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Hepatoprotective Effects and Antioxidant Role of Scutia Myrtina on Paracetamol Induced Hepatotoxicity in Rats
Ramanathan sambath kumar Dr, College of pharmacy, Seventh of April university, Zawia, Libya. Kupusamy Asokkumar Dr, College of Pharmacy, Seventh of April University, Zawia,Libya Nallasamy Venkateswara Murthy Dr, J.K.K.Natarajh College of Pharmacy, Tamil Nadu, India.

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Hepatoprotective Effects and Antioxidant Role of Scutia Myrtina on Paracetamol Induced Hepatotoxicity in Rats
Ramanathan sambath kumar Dr, Kupusamy Asokkumar Dr, and Nallasamy Venkateswara Murthy Dr

Abstract
The hepatoprotective effect and antioxidant role of ethanol extract of Scutia myrtina (EESM) was evaluated against paracetamol induced liver damage in rats. The degree of protection was measured by using biochemical parameters such as serum transaminase (SGPT and SGOT), alkaline phosphatase (ALP), bilirubin, total protein and uric acid. Further, the effects of the extract on lipid peroxidation (LPO), glutathione (GSH), Vitamin C and Vitamin E, superoxide dismutase (SOD), and catalase (CAT) were estimated. The ethanol extract of Scutia myrtina (EESM) (100 and 200 mg/kg) produced significant (P < 0.05) hepatoprotective effect by decreasing the activity of serum enzymes, bilirubin, and lipid peroxidation, while it significantly increased the levels of protein, uric acid, GSH, Vitamin C, Vitamin E, SOD and CAT (P < 0.05). The effects of EESM were comparable to that of standard drug Silymarin. The results indicate that EESM shows hepatoprotective effects on paracetamol-induced liver damage in rat this may due to antioxidant and free radical scavenging activity of EESM. KEYWORDS: Scutia myrtina, Hepatoprotective effects, Antioxidant, Paracetamol, Serum enzyme.

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1. Background Traditional use: Scutia myrtina (Burm.f.) Kurz (Rhamnaceae) is widely available in South India. It is commonly known as Chimat (Hindi), a prickly shrub found throughout the hotter parts of India, East Africa, Kenya, Tanzania, and South Africa. The aerial part of the plant was used for stomach problems, salpingitis. The root and leaves of the plant traditionally used as an antihelmintic (Kokwaro, et al 1976). In eastern Tanzania the root of this plant is used for the treatment of bilharzias, intestinal worms and fever (Chhabra et al., 1982). The leaves and root bark of the Scutia myrtina decoction is used for gonorrhea, bilharzias, and intestinal worms in Tanzania (Hedberg et al., 1983). The tribal peoples of Kolli Hills of Tamil Nadu India used the whole plant for the treatment of tumor, inflammation and liver disorders (Kritheka et al., 2008). Known pharmacological or biological activity: The alcohol extract of the aerial part of Scutia myrtina possess antiviral activity (Dhar et al., 1968). The root bark of the plant is used for fever and also the infusion of the plant is used to treat malaria. An alkaloid nitidine with potent antimalarial activity has been isolated from a Kenyan herbal remedy (Gakurju et al., 1995). Pervious report from our laboratory showed the anti-inflammatory and antimicrobial activity of petroleum ether and ethanol extracts of Scutia myrtina ((Kritheka et al., 2008). Rationale for the study: Scutia myrtina (Rhamnaceae) is a shrub traditionally used in the treatment of various infection and disease by the triple people in the different part of world and also has many pharmacological and biological activities as mentioned above but lack detailed reports on its hepatoprotective profile. Based on the previous reports, traditional usage and preliminary phytochemical analysis the ethanol extracts of Scutia myrtina (EEMS) was selected for the present study to demonstrate the hepatoprotective and antioxidant activities of Scutia myrtina on paracetamol induced hepatotoxicity in rats 2. Materials used in the study Methodology Raw herb: The whole plant Scutia myrtina Type of extract: Ethanol extract of Scutia myrtina Authentication: The plant material was taxonomically identified by the Botanical Survey of India, Coimbatore, Tamilnadu, India and the voucher specimen RRI/BNG/SMP-Prog/945 was retained in Natural product research laboratory, J.K.K.Nataraja College of Pharmacy, Tamilnadu, India, for future reference. Extraction: The entire plant of Scutia myrtina was dried under shade and then powdered with a mechanical grinder. The powder was passed through sieve

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number 40 and retained in sieve number 60 and stored in an airtight container for further use. The dried powder material of the plant (500g) was defatted with petroleum ether (60-80C) for 48 hours in soxhlet apparatus (yield 2.75% w/w). The defatted plant material thus obtained was further extracted with ethanol for 72 hours in the soxhlet. The solvent was removed by distillation under reduced pressure and the resulting semisolid mass was vacuum dried using rotatory flash evaporator to yield (7.45% w/w) a solid residue (ethanol extract). Different doses of EESM were prepared by dissolving a known weight of dried paste in 0.5% Carboxy methylcellulose (CMC). Characterization and standardization: Three new anthrone-anthraquinones, scutianthraquinones A, B and C, one new bisanthrone-anthraquinone, scutianthraquinone D, and the known anthraquinone, aloesaponarin I were isolated from ethanol extract of bark of Scutia myrtina (Yanpeng Hou et al., 2009). An alkaloid nitidine with potent antimalarial activity has been isolated from Scutia myrtina (Gakurju et al., 1995). Two new perylenequinones, scutiaquinone A and scutiaquinone B, have been isolated from a methanol extract of the roots of Scutia myrtina (Ayers et al., 2007). Experimental Animals: Studies were carried out using male Wistar albino rats weighing 150180 g were used. They were obtained from the animal house J.K.K.Nataraja College of Pharmacy, Tamilnadu, India. The animals were grouped and housed in polyacrylic cages with not more than six animals per cage and maintained under standard laboratory conditions (temperature 25 + 2oC) with dark and light cycle (14/10 h). They were allowed free access to standard dry pellet diet and water ad libitum. The rats were acclimatized to laboratory condition for 10 days before commencement of experiment. All procedures described were reviewed and approved by the institutional animal ethical committee. Experimental design: Paracetamol induced liver damage in rats Healthy albino rats were divided into 5 groups of 6 animals in each. Group 1, which served as normal, received normal saline (0.9 % w/v, NaCl), 5 ml/kg. Group 2 received paracetamol (500 mg/kg p.o) and standard drug Silymarin (25 mg/kg) once daily for 10 days (control). Group 3 received paracetamol (500 mg/kg p.o) once daily for 10 days (control). Group, 4, and 5 received paracetamol (500 mg/kg, p.o.) and EESM (100 and 200 mg/kg) respectively for 10 days (Subramoniam et al., 1998). The biochemical parameters were determined after 18 h fasting of the last dose.

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3. Biological activity examined and name of analyses: Estimation of serum and liver biochemical parameters The blood samples were collected from rats and allowed to clot for 45 min at room temperature. Serum was separated by centrifugation at 2500 rpm at 30C for 15 min and used for the estimation of SGPT, SGOT, SALP, serum bilirubin, total protein and uric acid by using automatic chemistry analyzer (Hitachi model 912 Rochi). The rats were sacrificed and their livers excised, rinsed in ice cold normal saline followed by 0.15 M Tris-HCl (pH 7.4) blotted dry and weighed. A 10 w/v % of homogenate was prepared in 0.15 M Tris-HCl buffer and processed for the estimation of LPO by Ohkawa et al. (1973). A part of homogenate after precipitating proteins with Trichloroacetic acid (TCA) was used for estimation of glutathione by the method of Ellman, (1959). Vitamin E by Quaife and Dju, (1948), with slight modification by Baker and Frank, (1951) and vitamin C by Omaye et al., (1979) were also estimated. The rest of the homogenate was centrifuged at 15000 rpm for 15 min at 4C. The supernatant thus obtained was used for the estimation of SOD by the method of Kakkar et al., (1984) and CAT activities were measured by the method of Aebi, (1974). Statistical analysis: The experimental results were expressed as mean S.E.M. Data were assessed by using statistical package for social science (SPSS) version 10.0 using ANOVA followed by Dennetts test. P value of < 0.05 was considered as statistically significant. 4. Research findings: The effect of EESM on serum transaminase, alkaline phosphatase, bilirubin, uric acid and total protein levels in paracetamol induced liver damage rats are summarized in figure1 and 2. There was a significant increase in the SGPT and SGOT in paracetamol control group as compared with normal group (P < 0.01). Administration of EESM at different doses (100 and 200 mg/kg. b.w.) to paracetamol induced rats the levels of SGPT and SGOT were reduced by 47.84, 83.01%, and 49.95 and 82.44%, respectively, in comparison to the paracetamol control group (P < 0.05). The levels of SALP serum bilirubin in the liver of the paracetamol control group increased in comparison with the normal group (P < 0.01). After administration of EESM at the dose of 100 and 200 mg/kg. b.w. decreases the levels of SALP and bilirubin by 55.44, 77.45% and 30.63 and 79.27% respectively, as compared to that of the paracetamol control group (P < 0.05). The uric acid and protein levels in paracetamol control group decreases in comparison with the normal group (P < 0.01). Treatment with EESM at doses of 100 and 200 mg/kg. b.w. increases the levels of uric acid and protein by 39.94, 92.95% and 25.50 and 91.19%, respectively, when compared to that of

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the paracetamol control (P < 0.05). Silymarin almost completely restored the serum transaminase, alkaline phosphatase, bilirubin, uric acid and total protein level in paracetamol treated groups to the normal level. The effects of EESM on rat liver lipid peroxidation, glutathione, and antioxidant enzyme levels are shown figure 3, 4, 5 and 6. Lipid peroxidation levels (expressed in term of malondialdehyde (MDA formation)) are significantly high in paracetamol control rats compared with the normal rats (P < 0.01). Treatment with EESM (100 and 200 mg/kg) significantly decreases the levels of MDA levels by 28.35 and 79.10% (P < 0.05) and bring them near to normal level (figure 3). The effect of EESM on glutathione content, vitamin C and vitamin E levels in the liver is shown in (figure 4 and 5). GSH level in normal group was measured to be higher than in paracetamol control group (P < 0.01). GSH level of EESM 100 and 200 mg/kg groups were increased by 46.40 and 88.88% respectively as compared to paracetamol control group (p < 0.05). The levels of Vitamin C and vitamin E in the liver of paracetamol control group decreased in comparison with the normal group (p < 0.01). After administration of EESM at the dose of 100 and 200 mg/kg., b.w. increased the levels of vitamin C and vitamin E by 42.25, 68.79% and 31.77% 93.35% respectively, as compared to that of the paracetamol control group (p < 0.05). Silymarin almost completely restored the glutathione, vitamin C and vitamin E level in paracetamol treated groups to the normal level. The SOD, and CAT levels were significantly increased (P < 0.05) in paracetamol control group (figure 6). Treatment with EESM (100 and 200 mg/kg) the SOD, and CAT levels were significantly decrease by 44.93, 87.09% and 42.95, 87.74% respectively, as compared to that of the paracetamol control group (p < 0.05). 5. Conclusions: Paracetamol (Acetaminophen) is a widely used antipyretic analgesic produces acute liver damage if overdoses are consumed. The hepatotoxicity of paracetamol has been attributed to the formation of toxic metabolites. Treatment with EESM decreases the levels of SGPT, SGOT, SALP, bilirubin and uric acid towards the respective normal value that is an indication of stabilization of plasma membrane as well as repair of hepatic tissue damage caused by paracetamol. Elevations in the levels of end products of lipid peroxidation and decrease in the levels of GSH, Vitamin C and Vitamin E, SOD, and catalase CAT in liver of rat treated with paracetamol were observed. Treatment with EESM significantly reversed these changes. Hence it may be possible that the mechanism of hepatoprotection of EESM is due to its antioxidant effect. From the results of this study it can be concluded that EESM has a potent hepatoprotective action upon paracetamol induced liver damage in rats. Our results show that the hepatoprotective and antioxidant effects of EESM may be

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due to its antioxidant and free radical scavenging properties. Therefore, the hepatoprotective effect of EESM as claimed by the traditional system has a sound scientific basis. 6. Significance, applications and implications: From the present study it can be inferred that the ethanol extracts of Scutia myrtina possessed significant hepatproctive and antioxidant activity in rat against paracetamol induced hepatotoxicity. Hepatoprotective and antioxidant effects of EESM may be due to its antioxidant and free radical scavenging properties. Therefore, number of scientific reports indicated certain flavonoids, alkaloids, triterpenoids and steroids have protective effect on liver due to its antioxidant properties. Presence of those compounds in EESM may be responsible for the protective effect on paracetamol induced liver damage in rats. The results can substantiate the traditional and folkloric uses of Scutia myrtina in Kolli Hills of Tamilnadu, India. Purification of the extracts and further studies can reveal the exact mechanisms and constituents behind the hepatoprotective and antioxidant activities of ethanol extract of Scutia myrtina. 7. References: Aebi H. Catalase 1974. In: Bergmeyer HV, ed, Methods in Enzymatic Analysis. Vol 2, New york, Academic Press, pp. 674-684. Ayers S, Zink DL, Mohn K, Powell JS, Brown CM, Murphy T, Brand R, Pretorius S, Stevenson D, Thompson D, Singh SB. Scutiaquinones A and B, Perylenequinones from the Roots of Scutia myrtina with Anthelmintic Activity. J Nat Prod 2007, 70: 425427. Baker AF and Frank. Estimation of vitamin E in tissues. 1951. In: G. Bollinger (ed.) Dunnschicht. G Chromaeographic in Laboratorium Handbuch. Springer Verlag, Berlin pp. 4152. Chhabra SC, Mahunnah RLA, Mshiu EN. 1991. Plants used in Traditional Medicine in Eastern Tanzania. V. Angiosperms (Passifloraceae to Sapindaceae). J Ethnopharmacol 7: 143-157. Dhar ML, Dhar, MM, Dhawan BN, Mehrotra BN, Roy C. 1968. Screening of Indian medicinal plant for biological activity. Ind J Expt Biol 6: 232-247 Ellman, GL, 1959.Tissue sulphydryl groups. Arch Biochem Biophys. 82: 70-77. Gakunju DMN, Mberu EK, Dossaji SF, Gray AI, Waigh RD, Waterman PG, Watkins WM. 1995. Potent antimalarial activity of the Alkaloid Nitidine, Isolated from a Kenyan herbal remedy. Antimicrob Agents Chimother. 39 2606-2609. Hedberg I, Hedberg O, Mashigeni KE, Mshiu, EN, Samuelsson G, 1983. Inventory of plants usedin traditional medicine in Tanzania, part III Plants the families Papilionaceae-Vitaceae. J Ethnopharmacol 9: 237-260.

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Kakkar P, Das B, Viswanathan PN. 1984. A modified spectrophotometric assay of superoxide dismutase. Ind J Biochem Biophys 21: 131-132. Kritheka N, Sambath Kumar R, Senthil V, Venkateswara murthy N, Shanmuga sundram R, Perumal P. 2008. Anti-inflammatory and antimicrobial activities of petroleum ether and ethanol extracts of Scutia myrtina. Journal of Oriental Pharm and Exp Med 8:400-407. Kokwaro JO. 1976. Medicinal plants of East Africa. East Africa literature bureau, Nairobi. Ohkawa H, Onishi N, Yagi K, 1979. Assay for lipid peroxidation in animal tissue by thiobarbituric acid reaction. Anal Biochem 95: 351-358. Omaye ST, Turun ball TD, Sauberlich HE. 1979. Selected method for the determination of ascorbic acid in animal cells, tissues and fluids. Meth Enzymol 62: 1-11. Quaife MC and Dju MY 1948. Chemical estimation of vitamin E in tissue and tocopherol content of normal tissues. J Biol Chem 180: 263272. Subramoniam A, Evans DA, Rajasakharan S, Pushpangadan. 1998. Hepatoprotective activity of Trichopus Zeylanicus extracts against paracetamol-induced damage in rats. Ind J Expt Biol 36: 385-389 Yanpeng Hou, Shugeng Cao, Peggy J. Brodie, Martin W. Callmander, Fidisoa Ratovoson, Etienne Rakotobe, Vincent E. Rasamison, Michel Ratsimbason John N. Alumasa, Paul D. Roepe, and David G. I. Kingston. 2009. Antiproliferative and antimalarial anthraquinones of Scutia myrtina from the Madagascar forest, Bioorg Med Chem 17: 28712876.

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